This proposal describes complementary genetic and molecular biological approaches to the study of aromatic amino acid biosynthesis and regulation in the flowering plant Arabidopsis thaliana. These experiments focus on the expression and physiological functions of DHS1 and DHS2, two Arabidopsis catalyzes the first committed step in aromatic amino acid biosynthesis. The expression of DHS1 is induced by wounding and pathogenic attack, whereas the expression of DHS2 is unaffected by these treatments. The induction of DHS1 by environmental stresses suggests that it has a direct role in accommodating aromatic secondary metabolism. A diverse array of secondary metabolic compounds that are derived from aromatic amino acids are critical to normal development and defense responses in plants. These compounds include UV-absorbing pigments, vitamins, structural polymers, antimicrobial toxins, and alkaloids: some also have important medical uses, such as morphine, codeine, vinblastine and vincristine. The production of aromatic secondary compounds in specific cell-types is controlled by complex developmental and environmental signals, and appears to involve the coordinate regulation of DHS1 and other genes involved in aromatic amino acid biosynthesis and secondary metabolism. The differential expression of DHS1 and DHS2 also suggest that these duplicate genes are regulated independently to accommodate distinct physiological requirements, such as secondary metabolism and protein synthesis. This proposal is designed to investigate the basis for the differential regulation of DHS1 and DHS2, and to determine the physiological functions of each gene. Specially, the cell type-specific expression pattern of DHS1 and DHS2 will be studied in transgenic plants expressing promoter- reporter gene fusions. The physiological roles played by both genes will be investigated using transgenic plants that over-express sense or antisense RNAs derive from DHS1 or DHS2. In a separate approach, genetic screens will be conducted at the whole plant level to obtain mutations in DHS1 and DHS2 function. These mutations will be identified by screening for changes in DAHP synthase activity among (1) previously identified suppressor of a existing Arabidopsis aromatic amino acid pathway mutation, and (2) a collection of embryo-lethal Arabidopsis mutants. In addition, transgenic Arabidopsis strains expressing a selectable marker gene will be used to obtain mutations in regulatory genes controlling DHS1 and DHS2 expression. This work will offer new insights into the genetic mechanisms by which biosynthetic pathways are regulated to accommodate complex metabolic requirements in multicellular organisms. These regulatory mechanisms may differ fundamentally from those described in microorganisms, such as transcriptional attenuation or GCN4-mediated general control.